The invention is directed toward an analytical method to determine the concentration of the free analyte fraction in a sample. More particularly, the method encompasses the use of affinity chromatography to determine the concentration of the free analyte fraction in a sample.
Many drugs, hormones, and toxins exist in two distinct forms as they pass through the blood stream: 1) a fraction that is non-covalently bound to proteins or other blood components and 2) a fraction that is non-bound, or free, in solution. The free and bound fractions are present in a dynamic state, in which solutes in one state are continually exchanging with those in the other. Accordingly, in biological systems this process is constant and an equilibrium is formed between the free and bound fractions.
It has long been hypothesized that the free fraction of such substances is the biologically-active form, since it is this form which crosses cell membranes and interacts with cell receptors or other target ligands. Because the free fraction is the biologically-active form, this makes analysis of free fractions of these substances of particular interest in clinical chemistry and pharmaceutical science as a means for controlling and studying their effects within the body.
For many substances it is possible to use their total concentrations in blood or serum as estimates of their free levels by assuming there is a constant relationship between these two types of values. However, there are numerous situations where this approach does not provide meaningful, or even remotely accurate information. For example, after surgery, during malnutrition or pregnancy, and in various disease states (e.g., cancer, renal failure or liver disease) there can be a large fluctuation in the concentration of binding proteins present in blood. This can shift the equilibrium between these proteins and drugs that bind to them and concomitantly cause a change in a drug""s free fraction even though its total concentration remains unaffected. Similar shifts in drug-protein binding can occur with age (e.g., in newborns or the elderly) and in situations where several drugs and/or endogenous agents compete for the same binding proteins. A drug with a high total concentration versus its binding proteins also creates problems when trying to estimate the free fraction based upon total protein concentrations since this may result in a non-linear relationship between the drug""s total and free levels.
Although several analytical methods have been developed in an attempt to determine the free fraction, all of these methods are plagued with inherent inaccuracies or are lengthy and tedious to perform. Examples of these methods include equilibrium dialysis, ultrafiltration and the use of natural filtrates, such as tears or saliva. A major problem with these techniques is that the analysis often involves the use of an additional binding reagent or separation process that interacts with the free or bound fraction and causes the equilibrium between these fractions to be altered. For example, techniques with long analysis times, on the order of several seconds, results in bias in the measurement process because it allows the release of a significant amount of solutes from the bound fraction which is then detected with the original free fraction. The end result is an error in the apparent concentration of free fraction that is measured. In addition, many of these techniques suffer from non-specific interactions (e.g., binding of drugs to dialysis or filtration membranes), and are limited to only certain types of analytes (as is the case with natural filtrates).
Accordingly, a need exists to determine the free fraction without impacting the equilibrium between the free and bound fractions of the solutes. Equally, a need exists for a method that is highly specific and can be employed to determine the free fraction of a vast number of different substances.
Among the several aspects of the invention therefore, is provided a method to determine the concentration of a free analyte fraction in a sample, the sample comprising a bound analyte fraction and the free analyte fraction, the method comprising applying the sample to an affinity column wherein the column separates the free analyte fraction from the sample in the millisecond time domain,detection of signal from the free analyte fraction separated from the sample, and determining the concentration of free analyte present in the sample.
In yet another aspect of the invention is provided a method to determine the concentration of a free analyte fraction in a sample, the sample comprising a bound analyte fraction and a free analyte fraction, the method comprising applying the sample to an affinity column, the column separating the sample into the free analyte fraction and the bound analyte fraction in the millisecond time domain wherein the free analyte fraction is adsorbed to the column and the bound analyte fraction passes through the column, applying a series of standards to the affinity column wherein each standard applied comprises a known concentration of the same free analyte present in the sample, the column separating each of the standards into a free fraction and a bound fraction in the millisecond time domain wherein the free fraction is adsorbed to the column and the bound fraction passes through the column, detection of signal from the free analyte fraction separated from the sample in (a) above, detection of signal from the free analyte fraction separated from the standard in (b) above, generating a calibration curve based upon the signal detected in (d) above, the curve comprising a graph of the concentration of free analyte present in each standard versus the signal detected for each concentration, and determining the concentration of the free analyte fraction present in the sample by comparing the signal detected in (c) above with the calibration curve in (e) above.
Yet a further aspect of the invention provides a method to determine the concentration of a free analyte fraction in a sample, the sample comprising a bound analyte fraction and a free analyte fraction, the method comprising applying the sample to an affinity column, the column separating the sample into the free analyte fraction and the bound analyte fraction in the millisecond time domain wherein the free analyte fraction is adsorbed to the column and the bound analyte fraction passes through the column, applying the sample set forth in (a) to an inert control column wherein the free analyte fraction does not adsorb to the column and a total analyte fraction comprising the free analyte fraction and the bound analyte fraction passes through the column, applying a series of standards to the affinity column wherein each standard applied comprises a known concentration of the same analyte present in the sample, the column separating each of the standards into a free fraction and a bound fraction in the millisecond time domain wherein the free fraction is adsorbed to the column and the bound fraction passes through the column, applying the same series of standards set forth in (c) to an inert control column wherein the free analyte fraction does not adsorb to the column and a total analyte fraction comprising the free analyte fraction and the bound analyte fraction passes through the column, detection of signal from the bound analyte fraction of the sample in (a) above, detection of signal from the total analyte fraction of the sample in (b) above, detection of signal from the bound fraction of the standard in (c) above, detection of signal from the total analyte fraction of the standard in (d) above, generating a calibration curve based upon the signal detected in (g) above, the curve comprising a graph of the bound concentration of analyte present in each standard versus the signal detected for each concentration, generating a calibration curve based upon the signal detected in (h) above, the curve comprising a graph of the total concentration of analyte present in each standard versus the signal detected for each concentration, determining the bound analyte fraction in the samples by comparing the signal in (e) above with the calibration curve in (i) above, determining the total analyte fraction in the samples by comparing the signal in (f) above with the calibration curve in (j) above, and determining the concentration of the free analyte fraction present in the sample by subtracting the bound analyte fraction in (k) above from the total analyte fraction in (1) above.
Still another aspect of the invention provides a method to determine the concentration of a free analyte fraction in a sample, the sample comprising a bound analyte fraction and the free analyte fraction, the method comprising applying the sample to an affinity column wherein the column separates the free analyte fraction from the sample in the millisecond time domain, detection of signal from the free analyte fraction separated from the sample and comparing the signal detected in (b) to a standard thereby determining the concentration of free analyte present in the sample.